Project 6 will use nanovectors to image and cotarget lethal prostate cancer and bone metastasis. This proposal builds on promising preliminary studies from our laboratory using bioluminescence to track normal prostate epithelial cells in transgenic mice and prostate-specific membrane antigen (PSMA) antibody conjugated nanoparticles to track prostate tumors in live animals. In this application, we further propose to develop and apply cutting edge nanotechnology to image and target bone metastasis, a lethal cancer phenotype in prostate, breast, and lung cancer patients. The primary goal is to develop multifunctional reagents to image, target and monitor the response of prostate cancer cells in cell culture and animal models. We also propose to develop a single cell gap junction dye transfer assay to differentiate prostate cancer cells with varying degrees of malignancy. Four specific aims are proposed. Specific aim 1 will validate prostate cancer, bone stromal, and endothelial cell surface biomarkers in clinical human prostate cancer specimens for imaging and targeting. We will confirm the expression of cell surface protein biomarkers in bone metastatic specimens from rapid autopsies and develop a gap junction dye transfer assay for prostate cancer and prostate or bone stromal cells. Specific aim 2 will explore the use of cell surface biomarkers as a targeting module to develop molecular imaging methods for prostate cancer, bone stromal, and endothelial cells grown as 3-D chimeric prostate cancer organoids and in transgenic, in situ and xenograft mouse models. Quantitative aspects of the assay will be calibrated using cells tagged with fluorescent proteins, Bioluminescent imaging using firefly luciferase will be tested in cultured cells and mouse models. Similar protocols will be used to target prostate cancer, bone stromal, and endothelial cells as in specific aim 3. We will develop an imaging method to detect apoptotic cells using annexin V imaging. Heparin-based biodegradable paclitaxel conjugates will be synthesized and tested in cell culture and animal models. Specific aim 4 will construct multifunctional nanoparticles to simultaneously image, target, and monitor therapeutic responses of prostate cancer, bone and lymph node metastases in animal models. Ultimately, this project will develop novel methods and multifunctional reagents to image, target, and monitor therapeutic responses to treat men with prostate cancer bone and lymph node metastases.